Embodiments of the inventive concepts described herein relate to an ultra-high-speed 3-dimensional (3D) refractive index tomography and structured illumination microscopy system using a wavefront shaper and a method using the same, and more particularly, to a technology that simultaneously measures a 3D high resolution fluorescence image and a 3D refractive index stereoscopic image of a cell by using a single wavefront shaper.
Nowadays, methods used to optically measure an inner structure of a cell are a fluorescence image method that expresses a fluorescent protein in a specific structure of a cell and measures a fluorescence signal and a 3D refractive index tomography that measures a 3D refractive index distribution in the cell.
Without the label of the fluorescent protein, the 3D refractive index tomography is used which measures the reaction of a cell with respect to an incident wavefront and measures the 3D refractive index distribution in a cell. Since the measured refractive index value is linearly proportional to the concentration of protein being a major component in a cell, a biochemical property, such as the distribution of the concentration of protein, as well as a structural property in a cell may be obtained from the 3D refractive index distribution in the measured cell. In addition, since the label of a fluorescent protein is not used, the 3D refractive index tomography is noninvasively applied to a living cell. The 3D refractive index tomography may measure the reaction of a cell for a long time because photobleaching does not occur.
The 3D refractive index tomography using various kinds of wavefront shapers is provided to measure the 3D refractive index image of a cell (non-patent document 1). The 3D refractive index tomography makes a light from a coherent light source incident on a cell and measures a hologram of a transmitted light, which is diffracted from the cell, by using an interferometer.
In this case, the 3D refractive index tomography may measure a plurality of 2-dimensional (2D) holograms while an angle, at which a light enters the cell, is changed by using the wavefront shaper and may measure the 3D refractive index distribution of a cell by analyzing the measured hologram information through an algorithm.
Recently, it is possible to make the ultra-high-speed 3D optical diffraction tomography by using the digital micromirror device being a kind of wavefront shaper (non-patent document 2).
The optical diffraction tomography may obtain a protein distribution in a cell. However, it is difficult to distinguish a specific protein because the optical diffraction tomography is not a specific measure at the molecular level.
Furthermore, for the fluorescence image of a cell, a fluorescent protein is expressed in a specific organ (molecule) of the cell or dye is attached thereto. If an excitation light source enters the expressed fluorescence material, the fluorescent protein emits a fluorescence signal of another wavelength after absorbing the excitation light source. After the 3D optical diffraction tomography distinguishes an image of the inner structure of a cell through the fluorescence signal, it measures the distinguished image.
Recently, a structured illumination microscopy began to be applied to a method of increasing the resolution of a fluorescence image. The structured illumination microscopy is a method of obtaining an image of an ultra-high resolution, which exceeds the value of a diffraction limit by making an excitation light source incident on cell with a specific pattern and measuring a signal that is beyond an optically measurable range.
At first, after the structured illumination microscopy forms a pattern by allowing a light to pass through a diffraction lattice, it obtained an image by using the pattern (non-patent document 3). The structured illumination microscopy may measure an image by using various patterns while rotating and moving a lattice pattern and may obtain an image of a high resolution from an image of a low resolution through the algorithm. Afterward, the structured illumination microscopy may easily obtain the image of a high resolution through control of a pattern shape through the wavefront shaper.
Recently, a nonlinear structured illumination microscopy made it possible to improve the resolution (non-patent document 4). However, in the case of a fluorescence image, a process of dying a cell to express a fluorescent protein is invasive. If the fluorescence image is measured for a long time, the photobleaching occurs. In this case, it is impossible to measure the cell.